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Determination of some heterocyclic aromatic amines in soup cubes by ion-pair chromatography with coulometric electrode array detection
Analytica Chimica Acta 417 (2000) 77–83
Determination of some heterocyclic aromatic amines in soup cubes by ion-pair chromatography with coulometric electrode array detection Christian Krach, Gerhard Sontag∗ Institute for Analytical Chemistry, University of Vienna, Waehringer Strasse 38, A-1090 Vienna, Austria Received 18 January 2000; received in revised form 11 April 2000; accepted 11 April 2000
Abstract An analytical method based on ion-pair chromatography with coulometric electrode array detection was developed for determination of four heterocyclic aromatic amines (IQ, MeIQx, 4,8-DiMeIQx and 7,8-DiMeIQx). These compounds are eluted isocratically (960 ml of a solution containing 30 mM trichloroacetic acid and 50 mM sodium acetate adjusted with glacial acetic acid to pH 2.5 are mixed with 40 ml tetrahydrofuran) from a phenyl–hexyl phase and are detected in a coulometric array detector. The working potentials of the four cells are set at +350, +690, +160 and +10 mV against modified palladium electrodes. The detection limits (S/N=3) depend on the working potential and are found between 0.1 and 1.1 ng injected. In commercial soup cubes, IQ, MeIQx and 4,8-DiMeIQx are present in concentrations between 0.3 and 2.8 ng/g. The recovery (54.8–100.9%) is influenced by the food matrix. The limits of quantitation are between 0.2 and 2.3 ng/g (S/N=5). © 2000 Elsevier Science B.V. All rights reserved. Keywords: Heterocyclic aromatic amines; Commercial soup cubes; Phenyl–hexyl phase; Ion-pair chromatography; Coulometric electrode array detection
1. Introduction Various types of carcinogens are present in food as minor components. One group are food-born heterocyclic aromatic amines (HAAs). Since the first detection of some HAAs by Sugimura and coworkers [1], several classes of HAAs have been identified in a wide range of food. Their chemistry and formation, their occurrence in food and their biological activity was reviewed by Eisenbrand and Tang [2] and Jagerstad et al. [3].
∗ Corresponding author. Tel.: +43-1-4277-52303; fax: +43-1-427-9523. E-mail address: [email protected] (G. Sontag)
Because of the low concentration level of HAAs in a highly complex food matrix selective and sensitive chromatographic techniques are required for detection and quantification. Therefore, most widely used chromatographic techniques are HPLC with fluorescence [4–6], electrochemical detection [5,7–10] and in last time more emphasized HPLC with mass spectrometry [11–13], whereas the selectivity of HPLC–UV [4,6,14] is sometimes too low for determination of HAAs in complex sample matrices. Liquid chromatographic separation of basic compounds on silica based material, especially heterocyclic aromatic amines, often results in tailing peaks. The primary cause of this behaviour is related to the silanol content in the stationary phase, which interact with basic analytes. To reduce peak tailing of HAAs,
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base deactivated RP-C18- and RP-C8-materials were used, e.g. ODS-Hypersil C18 [11], Nucleosil C18 [7], Li-Chrospher 60-RP select B [12,14] and TSK-Gel ODS 80-TM [4–6,8,13]. Another way to avoid peak tailing is to use mobile phase pH of less than 3 [15]. Under these conditions, silanol groups are mostly uncharged and the amines are protonated. In that way, the ion exchange interaction between silanols and HAAs are minimized. The aim of this work was to test a new phenyl–hexyl silica bonded phase for separation of HAAs and to improve the selectivity of detection of the electroactive HAAs in food by coulometric electrode array detection.
2. Experimental 2.1. Chemicals and standard solutions Heterocyclic aromatic amines under investigation were 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-tri-methylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f] quinoxaline (7,8-DiMeIQx) and the internal standard (IS) 2-amino-3,4,7,8-tetra-methylimidazo[4,5-f]quinoxaline (4,7,8-TriMeIQx), purchased from Toronto Research Chemicals (Toronto, Canada). Stock solutions of IQ (42 mg/l), MeIQx (22 mg/l), 4,8-DiMeIQx (44 mg/l) and 7,8-DiMeIQx (28 mg/l) were perpared in methanol. These solutions are further diluted with eluent and mixed with internal standard (4,7,8-TriMeIQx) solution to the appropriate concentration. The internal standard concentration was held constant at 270 ng/ml. A buffer stock solution, containing 200 mmol/l trichloroacetic acid (TCA) and 333 mmol/l sodium acetate trihydrate, adjusted with glacial acetic acid to pH 2.5, was prepared. Diatomaceous earth (Extrelut) were provided by Merck (Darmstadt, Germany), Isolute PRS SPE columns (1 g/3 ml) were from International Sorbent Technology Ltd. (Mid Glamorgan, UK). Solvents and chemicals used, were analytical-reagent grade (Merck, Darmstadt, Germany), UHQ-water was delivered by EASYpure LF Barnstead/Thermolyne, Dubuque, IO, USA).
2.2. Instrumentation Liquid chromatography was performed using a Merck Hitachi System D-6000, consisting of an AS-2000A autosampler adapted with an injection valve: Rheodyne 7000 E with a 40 l sample loop (Rheodyne, Cotati, CA, USA) and a L-6200 Intelligent Pump (Hitachi, Tokyo, Japan) equipped with a Luna phenyl–hexyl column, 250 mm×2.0 mm i.d., particle size: 5 m (Phenomenex, Torrance, CA, USA) and a phase separation ODS-2 precolumn, 10 mm×2 mm i.d., particle size: 5 m (Upchurch Scientific, Oak Harbor, WA, USA). For electrochemical detection an ESA CoulArray (ESA, Chelmsford, MA, USA) equipped with a cell block (model 6210, ESA, Chelmsford, MA, USA) with four seriate coulometric working electrodes (porous graphite) were used, controlled by an IBM PC/AT compatible computer provided with an ESA chromatographic software (Version 1.003). 2.3. Chromatographic separation and coulometric electrode array detection A standard solution (IQ: 168 ng/ml, MeIQx: 176 ng/ml, 7,8-DiMeIQx: 112 ng/ml, 4,8-DiMeIQx: 176 ng/ml) was injected and the amines were separated on the phenyl–hexyl column at room temperature using following eluents at a flow rate of 0.23 ml/min. 2.3.1. Influence of TCA concentration on capacity factors Buffer stock solution, distilled water and THF with different buffer stock solution–distilled water ratios (v/v/v) at constant THF content [(5, 10, 15, 22.5): (91, 86, 81, 73.5): 4] were mixed. Ionic strength was kept constant at 120 mmol/l by adding 93, 87, 40 and 0 mmol/l sodium chloride. 2.3.2. Influence of pH on capacity factors Buffer stock solution, distilled water and THF (15:81:4) were mixed and pH 2.1, 2.4, 2.6, 2.8 and 3.0 of mobile phase was then adjusted with concentrated hydrochloric acid or 10 M sodium hydroxyde solution. 2.3.3. Influence of THF content on capacity factors Buffer stock solution, distilled water and THF with different distilled water–THF ratios at constant buffer
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concentration [15: (81, 80.5, 80, 79.5, 79): (4, 4.5, 5, 5.5, 6)] were mixed. The working potentials of the four cells (four channels) were adjusted to +350, +690, +160 and +10 mV against modified palladium reference electrodes.
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recoveries were calculated with regard to the standard deviations of the slopes of both linear regression lines.
3. Results and discussion 3.1. Chromatographic separation
2.4. Analytical procedure A modified sample preparation, based on the method proposed by Gross [16] was applied. The sample (soup cubes) was homogenized and 5 g were weighed in a centrifuge tube. For standard addition samples were spiked with 250, 500 and 1000 l standard solution (IQ: 84 ng/ml, MeIQx: 88 ng/ml, 7,8-DiMeIQx: 56 ng/ml, 4,8-DiMeIQx: 88 ng/ml). The HAAs were extracted two times with 1 M hydrochloric acid and the extract (20 ml) was centrifuged and filtered. After alkalization of the crude extract (pH 10.5) with 6 M sodium hydroxide, the solution was mixed with Extrelut until the aqueous phase was bonded. The sample loaded Extrelut was transfered into an empty glass column (200 mm×25 mm i.d.), a PRS column, prewetted with 2 ml dichloromethane:toluene (10:1), was coupled and the HAAs were extracted with 70 ml dichloromethane:toluene (10:1). After extraction, the PRS column was disconnected, dried by strong vacuum and washed with 15 ml 0.1 M hydrochloric acid and 1 ml purified water. Elution of the HAAs was performed with 10 ml methanol:ammonia (9:1). The elute was evaporated to dryness and the residue was redissolved in 250 l eluent, containing 270 ng/ml IS. Using standard addition method, analyses were done twice for three spiked and one unspiked sample. After chromatographic separation and detection signals of Channels 3 and 4 were evaluated. All calculations were based on peak areas. HAAs in samples were quantified by determining the negative x-coordinate intercepts of the linear regression lines of standard additions for Channels 3 and 4. Relative standard deviations (R.S.D.) were calculated with regard to the standard deviations of slopes and intercepts of those linear regression lines [17]. The recoveries of HAAs under investigation were estimated for Channels 3 and 4 by dividing the slopes of the linear regression lines of standard additions by the slope of linear regression lines of the standards [18]. R.S.D. of
Trihalogenated acids can be used due to their pairing agent property [19,20] for separation of small cationic substances on the phenyl–hexyl column. As trichloroacetic acid (pKa =0.62) is fully dissociated and IQ compounds (pKa =6.0–7.0) are protonated [7] in the investigated pH-range, the formation of ion-pairs can be assumed. Varying mobile phase pH at constant TCA concentration and THF content, only small changes of k-values of HAAs are observed (Fig. 1), which can be explained by pH-depending changes of the stationary phase [15]. To control retention and selectivity of HAAs, TCA concentration and THF content were changed. With increasing mobile phase TCA concentration at constant pH and THF content, HAAs were increasingly retarded (Fig. 2). This retention behavior is most consistent with solvophobic ionic interactions with the pairing agent that enhance reversed-phase partitioning [19]. As retention of HAAs strongly depends on THF content (Fig. 3), only small amounts of THF were necessary to elute substances. The lower limit of 4% organic modifier is necessary, to avoid stationary phase collapse, which happens to silanol based RP-materials, working with pure aqueous mobile phases [21].
Fig. 1. k-values as function of the mobile phase pH.
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Fig. 2. k-values as function of the mobile phase TCA concentration.
Analyzing real samples interferences from the matrix are often observed at the beginning of the chromatograms. To avoid peak overlapping of early eluted substances, a mobile phase with 30 mM TCA at pH 2.5 and 4% THF was chosen. 3.2. Coulometric array detection Amperometric [8–10,22–24] detectors were frequently applied to detect HAAs oxidatively at a constant working potential. Coulometric electrode array detection [25,26] offered the possibility to detect such compounds at various potentials simultaneously. Nutt [27] and Springer [28] found that in a coulometric dual electrode detector HAAs can be oxidized in the
Fig. 3. k-values as function of the mobile phase THF content.
Fig. 4. Electrode array chromatograms of HAA standard solution (IQ: 42 ng/ml, MeIQx: 44 ng/ml, 7,8-DiMeIQx: 28 ng/ml, 4,8-DiMeIQx: 44 ng/ml, IS: 4,7,8-TriMeIQx: 270 ng/ml). 1: IQ, 2: MeIQx, 3: 7,8-DiMeIQx, 4: 4,8-DiMeIQx, IS: 4,7,8-TriMeIQx, Channel 2: +690 mV, Channel 3: +160 mV, Channel 4: +10 mV (inverted signals of Channel 4 are shown).
first cell and the products formed can be reduced in the second cell. Obtaining positive (Channel 3) and negative (Channel 4) signals with the coulometric array detector after primary oxidation of HAAs in Channel 2 (+690 mV), it can be concluded, that the oxidation products can be oxidized at a lower potential (+160 mV) than the parent compounds and further reduced at +10 mV in the following cell (Fig. 4). These potentials (+160 mV, +10 mV) are in the limiting current range of the formed compounds. On the basis of electrochemical reaction mechanism of 2-aminopyridine [29], it can be concluded, that heterocyclic aromatic amines can be oxidized in a 1e− , 1H+ step forming radicals. The dimerization of two radical species to the corresponding dihydrazocompound and following oxidation in a 2e− , 2H+ step at lower potential than for the HAAs leading to the
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Table 1 Limits of detection (LOD) based on a S/N=3, relative standard deviations (%) for run-to-run precision (n=6) and day-to-day precision (3 days, n=6) of HAAs under investigation LOD (ng)
IQ MeIQx 7,8-DiMeIQx 4,8-DiMeIQx
Run-to-run precision
Day-to-day precision
Channel 2
Channel 3
Channel 4
Channel 3
Channel 4
Channel 3
Channel 4
0.10 0.10 0.12 0.18
0.10 0.11 0.14 0.74
0.16 0.42 0.28 1.11
3.2 3.3 4.0 3.5
4.3 3.0 3.2 4.5
5.2 5.1 5.0 5.4
5.7 5.8 5.4 5.2
azo-compounds as final products. These products can be reduced again to the dihydrazo-compounds at low negative potential. A linear relationship between the response (peak area) of Channels 2, 3, and 4 and the concentration of HAAs in the range under investigation (IQ: 5.25–168 ng/ml, MeIQx: 11.0–176 ng/ml, 7,8-DiMeIQx: 7.0–112 ng/ml, 4,8-DIMeIQx: 22.0–176 ng/ml)
was found. The correlation coefficients of the calibration functions were better than 0.997. Limits of detection (LODs) for a S/N=3 (Table 1) and R.S.D. of run-to-run precision as well as of day-to-day precision (within 3 days) were calculated for n=6 (cf. standard concentration, see Section 2.3). The amount of oxidation products formed in Channel 2, which can be oxidized in Channel 3 (+160 mV)
Fig. 5. Chromatograms of a standard solution (I) and soup cube B extract (II) detected at Channel 3 (+160 mV); 1: IQ, 2: MeIQx, 3: 7,8-DiMeIQx, 4: 4,8-DiMeIQx, IS: 4,7,8-TriMeIQx.
Fig. 6. Chromatograms of a standard solution (I) and soup cube B extract (II) detected at Channel 4 (+10 mV, inverted signals of Channel 4 are shown); 1: IQ, 2: MeIQx, 3: 7,8-DiMeIQx, 4: 4,8-DiMeIQx, IS: 4,7,8-TriMeIQx.
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Table 2 Concentrations, recoveries and limits of quantitation (LOQ) based on a S/N=5 of HAAs in Soup cubes A and B IQ
MeIQx
7,8-DiMeIQx
4,8-DiMeIQx
Ox (Channel 3)
Red (Channel 4)
Ox (Channel 3)
Red (Channel 4)
Ox (Channel 3)
(Channel 4) (Channel 4)
(Channel 3) (Channel 3)
(Channel 4) (Channel 4)
n.q.
n.d.
n.d.
n.d.
n.d.
0.9 74.8 3.2
0.3 81.1 5.8
0.5 79.2 3.4
1.5 65.7 4.7
2.3 75.3 8.1
1.0 6.7 0.9 81.6 4.9
n.d.
n.d.
0.3 91.9 5.9
0.5 89.5 6.0
2.3 8.4 1.5 98.0 7.0
2.8 7.0 2.3 100.9 4.4
Soup cube A c (ng/g) R.S.D. (%) LOQ (ng/g) R (%) R.S.D. (%)
n.d.
n.d.
0.2 61.7 7.4
0.3 54.8 8.9
0.3 8.5 0.2 74.1 3.5
Soup cube B c (ng/g) R.S.D. (%) LOQ (ng/g) R (%) R.S.D. (%)
0.7 14.6 0.2 73.2 9.5
0.6 11.1 0.4 65.7 4.3
0.9 17.6 0.2 67.0 7.2
n.d.: Not detected; n.q.: not quantitated.
and reduced in Channel 4 (+10 mV) are especially for 4,8-DiMeIQx and 4,7,8-TriMeIQx significantly smaller than the amount of parent compounds. Therefore, smaller signals in Channels 3 and 4 are obtained but the low detector potentials (+160 and +10 mV) are responsible for a smaller noise than in Channel 2. This detection mode leads in some cases to higher detection limits (Table 1) but offers a better selectivity of detection.
of Channels 3 and 4 for each HAA with those of the standard compounds. The limits of quantitation (LOQs) were calculated for a S/N=5. Both channels were used to quantify the HAAs in the soup cubes and their content is summarized in Table 2 (Soup cubes A and B). The recoveries determined by standard addition method depend on the matrix of the sample.
3.3. Analysis of samples
4. Conclusions
To demonstrate the applicability of the method, two commercial soup cubes (A and B) consisting of salt, flavours, fat, starch, sugar, vegetables, spices, yeast extract and beef extract in different amounts were analyzed. After sample preparation, the extracts were chromatographed (Figs. 5 and 6). At the potential in the first cell (+350 mV), HAAs were not detected but some matrix compounds were oxidized and at least partially eliminated. Nevertheless, the relatively high potential of the second working electrode (+690 mV) makes it impossible to identify the HAAs because of overlapping with matrix compounds. Detecting the oxidation products of Channel 2 at Channel 3 (+160 mV, Fig. 5) and Channel 4 (+10 mV, Fig. 6) enhances the selectivity in such an extend, that HAAs under investigation could be identified and quantified. Peak purity was ascertained by matching signal ratios
It was shown that the coupling of ion pair liquid chromatography on the phenyl–hexyl phase with oxidative reductive coulometric array detection yields in contrast to the oxidative detection mode sufficient selectivity to determine HAAs in complex food matrix. The electrochemical oxidation of HAAs generates products which can be oxidised and reduced at very low potentials (+160 and +10 mV). This fact is responsible for low detector noise and high selectivity. Additionally the signals in both channels can be used to confirm the identity of resolved peaks and quantitative results. To optimize this kind of detection, research is necessary to elucidate the electrochemical reaction mechanism of HAAs. Small differences in the structure of quinoxalines, e.g. the methyl group in 4-position of 4,8-DiMeIQx and 4,7,8-TriMeIQx decreases the detector signal significantly.
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Although similar products were analyzed different coloured extracts and divergent recoveries were obtained. This shows that small differences in the food composition influence the recovery. Further investigations are necessary to achieve an efficient isolation and preconcentration of these compounds largely independent on the food matrix.
Acknowledgements We thank the ‘Fonds zur Förderung wissenschaftlicher Forschung’, Project No. 13171-CHE, for the financial support. References [1] T. Sugimura, T. Kawachi, M. Nagao, T. Yahagi, Y. Seino, T. Okamoto, K. Shudo, T. Kosuge, T. Tsuji, K. Wakabayashi, Y. Itaka, A. Itai, Proc. Jpn. Acad. 53 (1977) 58. [2] G. Eisenbrand, W. Tang, Toxicology 84 (1993) 1. [3] M. Jagerstad, K. Skog, P. Arvidson, A. Solyakov, Z. Lebensm, Unters. Forsch. A-Food Technol. 207 (1998) 419. [4] M.G. Knize, J.S. Felton, G.A. Gross, J. Chromatogr. 624 (1992) 253. [5] M.T. Galceran, P. Pais, L. Puignou, J. Chromatogr. A 719 (1996) 203. [6] M.G. Knize, C.P. Salmon, E.C. Hopmans, J.S. Felton, J. Chromatogr. A 763 (1997) 179. [7] Sp. Grivas, T. Nyhammar, Mutat. Res. 142 (1985) 5. [8] L. Rivera, M.J.C. Curto, P. Pais, M.T. Galceran, L. Puignou, J. Chromatogr. A 731 (1996) 85.
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[9] S.T. Billedeau, M.S. Bryant, C.L. Holder, LC–GC Int. 4 (1991) 38. [10] M.M.C. van Dyck, B. Rollmann, C. De Meester, J. Chromatogr. A 697 (1995) 377. [11] M.T. Galceran, E. Moyano, L. Puignou, P. Pais, J. Chromatogr. A 730 (1996) 185. [12] E. Richling, M. Herderich, P. Schreier, Chromatographia 42 (1996) 7. [13] P. Pais, E. Moyano, L. Puignou, M.T. Galceran, J. Chromatogr. A 778 (1997) 207. [14] G.A. Gross, G. Philippossian, H.H. Aeschbacher, Carcinogenesis 10 (1989) 1175. [15] J.W. Dolan, LC–GC Int. 12 (1999) 156. [16] G. Gross, Carcinogenesis 11 (1990) 1597. [17] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, Ellis Horwood PTR, Prentice-Hall, New York, 1993, p. 117. [18] W.S. Barnes, J.C. Maher, J.H. Weisburger, J. Agric. Food Chem. 31 (1983) 883. [19] W.A. MacCrehan, D. Shea, J. Chromatogr. 457 (1988) 111. [20] C. Krach, G. Sontag, S. Solar, Food Res. Int. 32 (1999) 43. [21] R.G. Wolcott, J.W. Dolan, LC–GC Int. 12 (1999) 260. [22] M. Takahashi, K. Wakabayashi, M. Nagao, M. Yamamoto, T. Masui, T. Goto, N. Kinae, I. Tomita, T. Sugimura, Carcinogenesis 6 (1985) 1195. [23] R. Schwarzenbach, D. Gubler, J. Chromatogr. 624 (1992) 491. [24] M. Murkovic, D. Steinberger, W. Pfannhauser, Z. Lebensm. Unters. Forsch. A 207 (1998) 477. [25] C. Bross, S. Springer, G. Sontag, Dtsch. Lebensm.-Rundsch. 93 (1997) 384. [26] G. Sontag, I. Bernwieser, C. Krach, Progress in HPLC–HPCE, Vol. 6, VSP, Utrecht, 1997, p. 75. [27] S. Nutt, Thesis, Westfälische-Wilhelms-Universität Münster, 1996. [28] S. Springer, Diploma Thesis, University of Vienna, 1996. [29] R.N. Goyal, A. Kumar, J. Electroanal. Chem. 242 (1988) 209.
Determination of some heterocyclic aromatic amines in soup cubes by ion-pair chromatography with coulometric electrode array detection Christian Krach, Gerhard Sontag∗ Institute for Analytical Chemistry, University of Vienna, Waehringer Strasse 38, A-1090 Vienna, Austria Received 18 January 2000; received in revised form 11 April 2000; accepted 11 April 2000
Abstract An analytical method based on ion-pair chromatography with coulometric electrode array detection was developed for determination of four heterocyclic aromatic amines (IQ, MeIQx, 4,8-DiMeIQx and 7,8-DiMeIQx). These compounds are eluted isocratically (960 ml of a solution containing 30 mM trichloroacetic acid and 50 mM sodium acetate adjusted with glacial acetic acid to pH 2.5 are mixed with 40 ml tetrahydrofuran) from a phenyl–hexyl phase and are detected in a coulometric array detector. The working potentials of the four cells are set at +350, +690, +160 and +10 mV against modified palladium electrodes. The detection limits (S/N=3) depend on the working potential and are found between 0.1 and 1.1 ng injected. In commercial soup cubes, IQ, MeIQx and 4,8-DiMeIQx are present in concentrations between 0.3 and 2.8 ng/g. The recovery (54.8–100.9%) is influenced by the food matrix. The limits of quantitation are between 0.2 and 2.3 ng/g (S/N=5). © 2000 Elsevier Science B.V. All rights reserved. Keywords: Heterocyclic aromatic amines; Commercial soup cubes; Phenyl–hexyl phase; Ion-pair chromatography; Coulometric electrode array detection
1. Introduction Various types of carcinogens are present in food as minor components. One group are food-born heterocyclic aromatic amines (HAAs). Since the first detection of some HAAs by Sugimura and coworkers [1], several classes of HAAs have been identified in a wide range of food. Their chemistry and formation, their occurrence in food and their biological activity was reviewed by Eisenbrand and Tang [2] and Jagerstad et al. [3].
∗ Corresponding author. Tel.: +43-1-4277-52303; fax: +43-1-427-9523. E-mail address: [email protected] (G. Sontag)
Because of the low concentration level of HAAs in a highly complex food matrix selective and sensitive chromatographic techniques are required for detection and quantification. Therefore, most widely used chromatographic techniques are HPLC with fluorescence [4–6], electrochemical detection [5,7–10] and in last time more emphasized HPLC with mass spectrometry [11–13], whereas the selectivity of HPLC–UV [4,6,14] is sometimes too low for determination of HAAs in complex sample matrices. Liquid chromatographic separation of basic compounds on silica based material, especially heterocyclic aromatic amines, often results in tailing peaks. The primary cause of this behaviour is related to the silanol content in the stationary phase, which interact with basic analytes. To reduce peak tailing of HAAs,
0003-2670/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 0 9 2 3 - 5
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C. Krach, G. Sontag / Analytica Chimica Acta 417 (2000) 77–83
base deactivated RP-C18- and RP-C8-materials were used, e.g. ODS-Hypersil C18 [11], Nucleosil C18 [7], Li-Chrospher 60-RP select B [12,14] and TSK-Gel ODS 80-TM [4–6,8,13]. Another way to avoid peak tailing is to use mobile phase pH of less than 3 [15]. Under these conditions, silanol groups are mostly uncharged and the amines are protonated. In that way, the ion exchange interaction between silanols and HAAs are minimized. The aim of this work was to test a new phenyl–hexyl silica bonded phase for separation of HAAs and to improve the selectivity of detection of the electroactive HAAs in food by coulometric electrode array detection.
2. Experimental 2.1. Chemicals and standard solutions Heterocyclic aromatic amines under investigation were 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-tri-methylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f] quinoxaline (7,8-DiMeIQx) and the internal standard (IS) 2-amino-3,4,7,8-tetra-methylimidazo[4,5-f]quinoxaline (4,7,8-TriMeIQx), purchased from Toronto Research Chemicals (Toronto, Canada). Stock solutions of IQ (42 mg/l), MeIQx (22 mg/l), 4,8-DiMeIQx (44 mg/l) and 7,8-DiMeIQx (28 mg/l) were perpared in methanol. These solutions are further diluted with eluent and mixed with internal standard (4,7,8-TriMeIQx) solution to the appropriate concentration. The internal standard concentration was held constant at 270 ng/ml. A buffer stock solution, containing 200 mmol/l trichloroacetic acid (TCA) and 333 mmol/l sodium acetate trihydrate, adjusted with glacial acetic acid to pH 2.5, was prepared. Diatomaceous earth (Extrelut) were provided by Merck (Darmstadt, Germany), Isolute PRS SPE columns (1 g/3 ml) were from International Sorbent Technology Ltd. (Mid Glamorgan, UK). Solvents and chemicals used, were analytical-reagent grade (Merck, Darmstadt, Germany), UHQ-water was delivered by EASYpure LF Barnstead/Thermolyne, Dubuque, IO, USA).
2.2. Instrumentation Liquid chromatography was performed using a Merck Hitachi System D-6000, consisting of an AS-2000A autosampler adapted with an injection valve: Rheodyne 7000 E with a 40 l sample loop (Rheodyne, Cotati, CA, USA) and a L-6200 Intelligent Pump (Hitachi, Tokyo, Japan) equipped with a Luna phenyl–hexyl column, 250 mm×2.0 mm i.d., particle size: 5 m (Phenomenex, Torrance, CA, USA) and a phase separation ODS-2 precolumn, 10 mm×2 mm i.d., particle size: 5 m (Upchurch Scientific, Oak Harbor, WA, USA). For electrochemical detection an ESA CoulArray (ESA, Chelmsford, MA, USA) equipped with a cell block (model 6210, ESA, Chelmsford, MA, USA) with four seriate coulometric working electrodes (porous graphite) were used, controlled by an IBM PC/AT compatible computer provided with an ESA chromatographic software (Version 1.003). 2.3. Chromatographic separation and coulometric electrode array detection A standard solution (IQ: 168 ng/ml, MeIQx: 176 ng/ml, 7,8-DiMeIQx: 112 ng/ml, 4,8-DiMeIQx: 176 ng/ml) was injected and the amines were separated on the phenyl–hexyl column at room temperature using following eluents at a flow rate of 0.23 ml/min. 2.3.1. Influence of TCA concentration on capacity factors Buffer stock solution, distilled water and THF with different buffer stock solution–distilled water ratios (v/v/v) at constant THF content [(5, 10, 15, 22.5): (91, 86, 81, 73.5): 4] were mixed. Ionic strength was kept constant at 120 mmol/l by adding 93, 87, 40 and 0 mmol/l sodium chloride. 2.3.2. Influence of pH on capacity factors Buffer stock solution, distilled water and THF (15:81:4) were mixed and pH 2.1, 2.4, 2.6, 2.8 and 3.0 of mobile phase was then adjusted with concentrated hydrochloric acid or 10 M sodium hydroxyde solution. 2.3.3. Influence of THF content on capacity factors Buffer stock solution, distilled water and THF with different distilled water–THF ratios at constant buffer
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concentration [15: (81, 80.5, 80, 79.5, 79): (4, 4.5, 5, 5.5, 6)] were mixed. The working potentials of the four cells (four channels) were adjusted to +350, +690, +160 and +10 mV against modified palladium reference electrodes.
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recoveries were calculated with regard to the standard deviations of the slopes of both linear regression lines.
3. Results and discussion 3.1. Chromatographic separation
2.4. Analytical procedure A modified sample preparation, based on the method proposed by Gross [16] was applied. The sample (soup cubes) was homogenized and 5 g were weighed in a centrifuge tube. For standard addition samples were spiked with 250, 500 and 1000 l standard solution (IQ: 84 ng/ml, MeIQx: 88 ng/ml, 7,8-DiMeIQx: 56 ng/ml, 4,8-DiMeIQx: 88 ng/ml). The HAAs were extracted two times with 1 M hydrochloric acid and the extract (20 ml) was centrifuged and filtered. After alkalization of the crude extract (pH 10.5) with 6 M sodium hydroxide, the solution was mixed with Extrelut until the aqueous phase was bonded. The sample loaded Extrelut was transfered into an empty glass column (200 mm×25 mm i.d.), a PRS column, prewetted with 2 ml dichloromethane:toluene (10:1), was coupled and the HAAs were extracted with 70 ml dichloromethane:toluene (10:1). After extraction, the PRS column was disconnected, dried by strong vacuum and washed with 15 ml 0.1 M hydrochloric acid and 1 ml purified water. Elution of the HAAs was performed with 10 ml methanol:ammonia (9:1). The elute was evaporated to dryness and the residue was redissolved in 250 l eluent, containing 270 ng/ml IS. Using standard addition method, analyses were done twice for three spiked and one unspiked sample. After chromatographic separation and detection signals of Channels 3 and 4 were evaluated. All calculations were based on peak areas. HAAs in samples were quantified by determining the negative x-coordinate intercepts of the linear regression lines of standard additions for Channels 3 and 4. Relative standard deviations (R.S.D.) were calculated with regard to the standard deviations of slopes and intercepts of those linear regression lines [17]. The recoveries of HAAs under investigation were estimated for Channels 3 and 4 by dividing the slopes of the linear regression lines of standard additions by the slope of linear regression lines of the standards [18]. R.S.D. of
Trihalogenated acids can be used due to their pairing agent property [19,20] for separation of small cationic substances on the phenyl–hexyl column. As trichloroacetic acid (pKa =0.62) is fully dissociated and IQ compounds (pKa =6.0–7.0) are protonated [7] in the investigated pH-range, the formation of ion-pairs can be assumed. Varying mobile phase pH at constant TCA concentration and THF content, only small changes of k-values of HAAs are observed (Fig. 1), which can be explained by pH-depending changes of the stationary phase [15]. To control retention and selectivity of HAAs, TCA concentration and THF content were changed. With increasing mobile phase TCA concentration at constant pH and THF content, HAAs were increasingly retarded (Fig. 2). This retention behavior is most consistent with solvophobic ionic interactions with the pairing agent that enhance reversed-phase partitioning [19]. As retention of HAAs strongly depends on THF content (Fig. 3), only small amounts of THF were necessary to elute substances. The lower limit of 4% organic modifier is necessary, to avoid stationary phase collapse, which happens to silanol based RP-materials, working with pure aqueous mobile phases [21].
Fig. 1. k-values as function of the mobile phase pH.
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Fig. 2. k-values as function of the mobile phase TCA concentration.
Analyzing real samples interferences from the matrix are often observed at the beginning of the chromatograms. To avoid peak overlapping of early eluted substances, a mobile phase with 30 mM TCA at pH 2.5 and 4% THF was chosen. 3.2. Coulometric array detection Amperometric [8–10,22–24] detectors were frequently applied to detect HAAs oxidatively at a constant working potential. Coulometric electrode array detection [25,26] offered the possibility to detect such compounds at various potentials simultaneously. Nutt [27] and Springer [28] found that in a coulometric dual electrode detector HAAs can be oxidized in the
Fig. 3. k-values as function of the mobile phase THF content.
Fig. 4. Electrode array chromatograms of HAA standard solution (IQ: 42 ng/ml, MeIQx: 44 ng/ml, 7,8-DiMeIQx: 28 ng/ml, 4,8-DiMeIQx: 44 ng/ml, IS: 4,7,8-TriMeIQx: 270 ng/ml). 1: IQ, 2: MeIQx, 3: 7,8-DiMeIQx, 4: 4,8-DiMeIQx, IS: 4,7,8-TriMeIQx, Channel 2: +690 mV, Channel 3: +160 mV, Channel 4: +10 mV (inverted signals of Channel 4 are shown).
first cell and the products formed can be reduced in the second cell. Obtaining positive (Channel 3) and negative (Channel 4) signals with the coulometric array detector after primary oxidation of HAAs in Channel 2 (+690 mV), it can be concluded, that the oxidation products can be oxidized at a lower potential (+160 mV) than the parent compounds and further reduced at +10 mV in the following cell (Fig. 4). These potentials (+160 mV, +10 mV) are in the limiting current range of the formed compounds. On the basis of electrochemical reaction mechanism of 2-aminopyridine [29], it can be concluded, that heterocyclic aromatic amines can be oxidized in a 1e− , 1H+ step forming radicals. The dimerization of two radical species to the corresponding dihydrazocompound and following oxidation in a 2e− , 2H+ step at lower potential than for the HAAs leading to the
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Table 1 Limits of detection (LOD) based on a S/N=3, relative standard deviations (%) for run-to-run precision (n=6) and day-to-day precision (3 days, n=6) of HAAs under investigation LOD (ng)
IQ MeIQx 7,8-DiMeIQx 4,8-DiMeIQx
Run-to-run precision
Day-to-day precision
Channel 2
Channel 3
Channel 4
Channel 3
Channel 4
Channel 3
Channel 4
0.10 0.10 0.12 0.18
0.10 0.11 0.14 0.74
0.16 0.42 0.28 1.11
3.2 3.3 4.0 3.5
4.3 3.0 3.2 4.5
5.2 5.1 5.0 5.4
5.7 5.8 5.4 5.2
azo-compounds as final products. These products can be reduced again to the dihydrazo-compounds at low negative potential. A linear relationship between the response (peak area) of Channels 2, 3, and 4 and the concentration of HAAs in the range under investigation (IQ: 5.25–168 ng/ml, MeIQx: 11.0–176 ng/ml, 7,8-DiMeIQx: 7.0–112 ng/ml, 4,8-DIMeIQx: 22.0–176 ng/ml)
was found. The correlation coefficients of the calibration functions were better than 0.997. Limits of detection (LODs) for a S/N=3 (Table 1) and R.S.D. of run-to-run precision as well as of day-to-day precision (within 3 days) were calculated for n=6 (cf. standard concentration, see Section 2.3). The amount of oxidation products formed in Channel 2, which can be oxidized in Channel 3 (+160 mV)
Fig. 5. Chromatograms of a standard solution (I) and soup cube B extract (II) detected at Channel 3 (+160 mV); 1: IQ, 2: MeIQx, 3: 7,8-DiMeIQx, 4: 4,8-DiMeIQx, IS: 4,7,8-TriMeIQx.
Fig. 6. Chromatograms of a standard solution (I) and soup cube B extract (II) detected at Channel 4 (+10 mV, inverted signals of Channel 4 are shown); 1: IQ, 2: MeIQx, 3: 7,8-DiMeIQx, 4: 4,8-DiMeIQx, IS: 4,7,8-TriMeIQx.
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Table 2 Concentrations, recoveries and limits of quantitation (LOQ) based on a S/N=5 of HAAs in Soup cubes A and B IQ
MeIQx
7,8-DiMeIQx
4,8-DiMeIQx
Ox (Channel 3)
Red (Channel 4)
Ox (Channel 3)
Red (Channel 4)
Ox (Channel 3)
(Channel 4) (Channel 4)
(Channel 3) (Channel 3)
(Channel 4) (Channel 4)
n.q.
n.d.
n.d.
n.d.
n.d.
0.9 74.8 3.2
0.3 81.1 5.8
0.5 79.2 3.4
1.5 65.7 4.7
2.3 75.3 8.1
1.0 6.7 0.9 81.6 4.9
n.d.
n.d.
0.3 91.9 5.9
0.5 89.5 6.0
2.3 8.4 1.5 98.0 7.0
2.8 7.0 2.3 100.9 4.4
Soup cube A c (ng/g) R.S.D. (%) LOQ (ng/g) R (%) R.S.D. (%)
n.d.
n.d.
0.2 61.7 7.4
0.3 54.8 8.9
0.3 8.5 0.2 74.1 3.5
Soup cube B c (ng/g) R.S.D. (%) LOQ (ng/g) R (%) R.S.D. (%)
0.7 14.6 0.2 73.2 9.5
0.6 11.1 0.4 65.7 4.3
0.9 17.6 0.2 67.0 7.2
n.d.: Not detected; n.q.: not quantitated.
and reduced in Channel 4 (+10 mV) are especially for 4,8-DiMeIQx and 4,7,8-TriMeIQx significantly smaller than the amount of parent compounds. Therefore, smaller signals in Channels 3 and 4 are obtained but the low detector potentials (+160 and +10 mV) are responsible for a smaller noise than in Channel 2. This detection mode leads in some cases to higher detection limits (Table 1) but offers a better selectivity of detection.
of Channels 3 and 4 for each HAA with those of the standard compounds. The limits of quantitation (LOQs) were calculated for a S/N=5. Both channels were used to quantify the HAAs in the soup cubes and their content is summarized in Table 2 (Soup cubes A and B). The recoveries determined by standard addition method depend on the matrix of the sample.
3.3. Analysis of samples
4. Conclusions
To demonstrate the applicability of the method, two commercial soup cubes (A and B) consisting of salt, flavours, fat, starch, sugar, vegetables, spices, yeast extract and beef extract in different amounts were analyzed. After sample preparation, the extracts were chromatographed (Figs. 5 and 6). At the potential in the first cell (+350 mV), HAAs were not detected but some matrix compounds were oxidized and at least partially eliminated. Nevertheless, the relatively high potential of the second working electrode (+690 mV) makes it impossible to identify the HAAs because of overlapping with matrix compounds. Detecting the oxidation products of Channel 2 at Channel 3 (+160 mV, Fig. 5) and Channel 4 (+10 mV, Fig. 6) enhances the selectivity in such an extend, that HAAs under investigation could be identified and quantified. Peak purity was ascertained by matching signal ratios
It was shown that the coupling of ion pair liquid chromatography on the phenyl–hexyl phase with oxidative reductive coulometric array detection yields in contrast to the oxidative detection mode sufficient selectivity to determine HAAs in complex food matrix. The electrochemical oxidation of HAAs generates products which can be oxidised and reduced at very low potentials (+160 and +10 mV). This fact is responsible for low detector noise and high selectivity. Additionally the signals in both channels can be used to confirm the identity of resolved peaks and quantitative results. To optimize this kind of detection, research is necessary to elucidate the electrochemical reaction mechanism of HAAs. Small differences in the structure of quinoxalines, e.g. the methyl group in 4-position of 4,8-DiMeIQx and 4,7,8-TriMeIQx decreases the detector signal significantly.
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Although similar products were analyzed different coloured extracts and divergent recoveries were obtained. This shows that small differences in the food composition influence the recovery. Further investigations are necessary to achieve an efficient isolation and preconcentration of these compounds largely independent on the food matrix.
Acknowledgements We thank the ‘Fonds zur Förderung wissenschaftlicher Forschung’, Project No. 13171-CHE, for the financial support. References [1] T. Sugimura, T. Kawachi, M. Nagao, T. Yahagi, Y. Seino, T. Okamoto, K. Shudo, T. Kosuge, T. Tsuji, K. Wakabayashi, Y. Itaka, A. Itai, Proc. Jpn. Acad. 53 (1977) 58. [2] G. Eisenbrand, W. Tang, Toxicology 84 (1993) 1. [3] M. Jagerstad, K. Skog, P. Arvidson, A. Solyakov, Z. Lebensm, Unters. Forsch. A-Food Technol. 207 (1998) 419. [4] M.G. Knize, J.S. Felton, G.A. Gross, J. Chromatogr. 624 (1992) 253. [5] M.T. Galceran, P. Pais, L. Puignou, J. Chromatogr. A 719 (1996) 203. [6] M.G. Knize, C.P. Salmon, E.C. Hopmans, J.S. Felton, J. Chromatogr. A 763 (1997) 179. [7] Sp. Grivas, T. Nyhammar, Mutat. Res. 142 (1985) 5. [8] L. Rivera, M.J.C. Curto, P. Pais, M.T. Galceran, L. Puignou, J. Chromatogr. A 731 (1996) 85.
83
[9] S.T. Billedeau, M.S. Bryant, C.L. Holder, LC–GC Int. 4 (1991) 38. [10] M.M.C. van Dyck, B. Rollmann, C. De Meester, J. Chromatogr. A 697 (1995) 377. [11] M.T. Galceran, E. Moyano, L. Puignou, P. Pais, J. Chromatogr. A 730 (1996) 185. [12] E. Richling, M. Herderich, P. Schreier, Chromatographia 42 (1996) 7. [13] P. Pais, E. Moyano, L. Puignou, M.T. Galceran, J. Chromatogr. A 778 (1997) 207. [14] G.A. Gross, G. Philippossian, H.H. Aeschbacher, Carcinogenesis 10 (1989) 1175. [15] J.W. Dolan, LC–GC Int. 12 (1999) 156. [16] G. Gross, Carcinogenesis 11 (1990) 1597. [17] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, Ellis Horwood PTR, Prentice-Hall, New York, 1993, p. 117. [18] W.S. Barnes, J.C. Maher, J.H. Weisburger, J. Agric. Food Chem. 31 (1983) 883. [19] W.A. MacCrehan, D. Shea, J. Chromatogr. 457 (1988) 111. [20] C. Krach, G. Sontag, S. Solar, Food Res. Int. 32 (1999) 43. [21] R.G. Wolcott, J.W. Dolan, LC–GC Int. 12 (1999) 260. [22] M. Takahashi, K. Wakabayashi, M. Nagao, M. Yamamoto, T. Masui, T. Goto, N. Kinae, I. Tomita, T. Sugimura, Carcinogenesis 6 (1985) 1195. [23] R. Schwarzenbach, D. Gubler, J. Chromatogr. 624 (1992) 491. [24] M. Murkovic, D. Steinberger, W. Pfannhauser, Z. Lebensm. Unters. Forsch. A 207 (1998) 477. [25] C. Bross, S. Springer, G. Sontag, Dtsch. Lebensm.-Rundsch. 93 (1997) 384. [26] G. Sontag, I. Bernwieser, C. Krach, Progress in HPLC–HPCE, Vol. 6, VSP, Utrecht, 1997, p. 75. [27] S. Nutt, Thesis, Westfälische-Wilhelms-Universität Münster, 1996. [28] S. Springer, Diploma Thesis, University of Vienna, 1996. [29] R.N. Goyal, A. Kumar, J. Electroanal. Chem. 242 (1988) 209.